The involvement of NLRP3 inflammasome in CUMS-induced AD-like pathological changes and related cognitive decline in mice

Abstract

Background: Numerous studies have found that inhibiting the expression of NLRP3 inflammasome can significantly improve depressive-like behaviors in mice, but the research on its effect on cognitive decline in depression and its mechanism is still lacking. This study aimed to elucidate the role of NLRP3 inflammasome in cognitive decline in depression and explore the common neuro-immunological mechanisms of depression and Alzheimer's disease (AD).

Methods: Male C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) for 5 weeks, treatment group was administered with the NLRP3 inhibitor MCC950 (10 mg/kg, i.p.), fluoxetine served as positive control. Then, the mice were assessed for cognitive behaviors and depression-like behaviors, and changes of microglia and neurons in hippocampus and levels of Aβ metabolic pathway and tau protein were measured. To explore the mechanism of NLRP3 activation on neurons, we performed in vitro studies using BV2 microglia and mouse primary neurons. Furthermore, we focused on the role of NLRP3 inflammasome in the function of neurons and the expression of AD pathological indicators.

Results: CUMS induced depressive-like behaviors and cognitive decline in mice, which could be reversed by inhibiting NLRP3 inflammasome. MCC950, a specific NLRP3 inhibitor, alleviated CUMS-induced neuron injury and AD-like pathological changes, including the abnormal expression of Aβ metabolic pathway and the hyper-phosphorylation of tau protein. LPS (1 μg/mL) + ATP (1 mM) treatment activated the expression of NLRP3 inflammasome and IL-1β in vitro. In vitro experiment also proved that inhibiting the expression of NLRP3 inflammasome in microglia can restore the Aβ metabolic pathway to normal, decrease neuronal tau protein phosphorylation and protect neurons.

Conclusions: Inhibition of NLRP3 inflammasome effectively alleviated CUMS-induced depressive-like behaviors and cognitive decline in mice, and inhibited the activation of AD physiological indicators.

Keywords: Alzheimer’s disease; Cognitive decline; Depression; NLRP3 inflammasome; Neuroinflammation.

Fig. 1

MCC950 improved CUMS-induced depressive-like behaviors and cognitive decline. A The sucrose preference in CUMS mice was lower than Control group, while MCC950 and fluoxetine could improve. B MCC950 and fluoxetine could reverse CUMS-induced prolonged tail suspension time. C MCC950 could reduce platform crossings numbers of CUMS mice, but fluoxetine could not. D MCC950, rather than fluoxetine, improved escape latency of CUMS mice. E, F The time of CUMS mice in the target quadrant was shortened, and both MCC950 and fluoxetine could relieve. The data were shown by mean and standard error (Mean ± SEM). (n = 9 for each group, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

MCC950 alleviated the decrease of immature neurons induced by CUMS. A Immunofluorescence was used to detect the expression and distribution of the target protein. B CUMS induced the decrease of DCX positive cells in hippocampus, and MCC950 could significantly improve. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01. Scale bar 100 μm)

Fig. 3

MCC950 reversed CUMS-induced decrease of Nissl staining positive cells. A Nissl staining in hippocampal CA1 and CA3 regions. B MCC950 and fluoxetine reversed CUMS induced decrease in the number of Nissl positive cells in CA1. C MCC950 and fluoxetine reversed CUMS induced decrease in the number of Nissl positive cells in CA3. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05. Scale bar 50 μm)

Fig. 4

MCC950 alleviated the activation of microglia induced by CUMS. A The expression of target protein was detected by Western Blotting. B There was no significant difference in GFAP expression among groups. C CUMS induced the up-regulation of Iba1 expression in hippocampus, while MCC950 and fluoxetine intervention could significantly improve. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)

Fig. 5

MCC950 inhibited CUMS-induced activation of NLRP3 inflammasome. A The expression of target protein was detected by Western Blotting. B MCC950 inhibited the up-regulation of NLRP3 expression induced by CUMS. C Both MCC950 and fluoxetine could significantly inhibit CUMS-induced activation of Caspase-1 p10. D CUMS induced up-regulation of IL-1β expression, and both MCC950 and fluoxetine could inhibit. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)

Fig. 6

MCC950 restored CUMS-induced activation of Aβ metabolic pathway. A The expression of target protein was detected by Western Blotting. B Both MCC950 and fluoxetine could inhibit CUMS induced up-regulation of APP. C Both MCC950 and fluoxetine could relieve the increased expression of BACE1 induced by CUMS. D CUMS induced down-regulation of IDE, which could be improved by MCC950 and fluoxetine intervention. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)

Fig. 7

MCC950 inhibited the increase of tau protein phosphorylation induced by CUMS. A The expression of target protein was detected by Western Blotting. B Both MCC950 and fluoxetine could alleviate CUMS induced the phosphorylation of tau protein at Ser396 site. C Both MCC950 and fluoxetine could alleviate CUMS induced the phosphorylation of tau protein at Ser202 site. D There was no significant difference in Tau5 protein among the groups. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)

Fig. 8

MCC950 reduced CUMS-induced activation of tau protein phosphorylation-related protein kinase. A The expression of target protein was detected by Western Blotting. B MCC950 and fluoxetine could improve CUMS induced the phosphorylation level of GSK-3β at Ser9 site. C CUMS significantly increased the phosphorylation level of ERK1/2 at Thr202/Tyr204 site, and both MCC950 and fluoxetine could decrease. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)

Fig. 9

MCC950 ameliorated the activation of NLRP3 inflammasome in BV2 cells. A The expression of target protein was detected by Western Blotting. B MCC950 inhibited the up-regulation of NLRP3 induced by LPS + ATP. C MCC950 inhibited the activation of Caspase-1 p10 induced by LPS + ATP. D MCC950 alleviated the up-regulation of IL-1β induced by LPS + ATP. The data were shown by mean and standard error (Mean ± SEM). (n = 4 for each group, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 10

MCC950 alleviated the activation of Aβ metabolic pathway in BV2 cells. A The expression of target protein was detected by Western Blotting. B MCC950 inhibited the up-regulation of APP in BV2 cells induced by LPS + ATP. C MCC950 ameliorated the up-regulation of BACE1 induced by LPS + ATP. D MCC950 improved the down-regulation of IDE induced by LPS + ATP. The data were shown by mean and standard error (Mean ± SEM). (n = 4 for each group, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 11

MCC950 reduced the phosphorylation level of tau protein. A The expression of target protein was detected by Western Blotting. B The secretion of BV2 cells activated by LPS + ATP induced the up-regulation of the phosphorylation level of tau protein in Ser396 site, and MCC950 could inhibit it. C The secretion of BV2 cells activated by LPS + ATP had no influence on the expression of total tau protein. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05)

Fig. 12

MCC950 reduced the activation of protein kinase related to tau protein phosphorylation. A The expression of target protein was detected by Western Blotting. B MCC950 inhibited the down-regulation of GSK-3β phosphorylation in primary neurons induced by the secretion of BV2 cells activated by LPS + ATP. C The secretion of BV2 cells activated by LPS + ATP induced the up-regulation of ERK1/2 phosphorylation in primary neurons, and the intervention of MCC950 could alleviate. The data were shown by mean and standard error (Mean ± SEM). (n = 3 for each group, *P < 0.05, **P < 0.01)